Antibodies find many applications in science and medicine. It is fairly straightforward to generate new antibodies against a target. For most applications antibodies are produced by so-called hybridoma cell lines that result from the fusion of an antibody producing B-cell with an immortalized cell line. Such hybridoma cells can easily be cultured and the antibody can be harvested from the culture supernatant. Another method for the production of antibodies is the harvesting from serum of immunized animals. Technology for the breeding of farm-animals is widespread and farm-animal housing is relatively cheap.
The production of immunogen-specific antibodies in mammary secretion products of farm animals has proven feasible some decades ago already. Initially, best results were obtained in the colostrum, i.e. the first lacteal fluid that is produced by the female following birth of a young. Milk produced by the female following the colostrial stage is called herein mature milk. Colostrum is quite a unique product that arises from a distinct physiological and functional state of the mammary gland. In ruminants, the principal compositional difference between colostrum and mature milk is the very high content of colostral immunoglobulin, 80-90% of which are of the IgG class. The antibody levels in mature milk are in principle lower (approximately an order of magnitude) then those that can be achieved in colostrum (Hodgkinson et al., WO 98/54226; Hastings, U.S. Pat. No. 5,017,372). Milk-derived antigen-specific antibodies employed in most clinical and preclinical studies therefore initially were in fact colostrum-derived and belonged predominantly to the IgG class (Tollemar et al., Bone Marrow Transpl. 23: 283-290 (1999); Bostwick et al., U.S. Pat. No. 5,773,000; Cordle et al., U.S. Pat. No. 5,260,057).
Immunoglobulin A (IgA) is an antibody playing a critical role in mucosal immunity. IgA is found in secretions in a specific form, which is referred to as S-IgA, comprising dimers of IgA monomers, linked by the so-called J-chain and further comprising the so-called secretory component, having a molecular weight of approximately 435 kDa. In its secretory form, it is the main immunoglobulin in mucous secretions, including tears, saliva, human colostrum/mature milk, gastro-intestinal juice, vaginal fluid and secretions from the prostate and respiratory epithelium. As such they can be found in the mucosal areas from the gastro-intestinal tract, respiratory tract, urogenital tract and oral/nasal cavity, and act to prevent colonization by pathogens. Secretory IgA can survive in harsh environments such as the digestive and respiratory tracts, to provide protection against microbes that multiply in body secretions. These properties make S-IgA the preferred immunoglobulin for application in products for improving and/or maintaining health, especially for treating and/or preventing infection and/or inflammation of mucosal surfaces, such as the gastro-intestinal mucosa and the mucosa of the respiratory tract but also of the skin. Examples of such products include enteral formulas, e.g. infant formulas, clinical nutrition, functional foods and nutraceuticals; pharmaceutical preparations, skin preparations, and aerosol preparations. It is therefore not surprising that a lot of effort has also been invested in producing increased levels of (secretory) IgA in ruminant milk.
U.S. Pat. No. 6,974,573 discusses a process of hyperimmunizing a farm-animal for an antigen via a mucosal passage or the airway and subsequently administering the antigen through a mammary gland or supramammary lymph node. It discusses using the milk so obtained directly or further processing for purifying the antigen-specific (IgA) antibodies.
Filtration fractionation of complex compositions, such as milk, to yield immunoglobulin-rich fractions has been described in the art, although the vast majority of such prior art disclosures concerns IgG isolation, which is not surprising given the fact that this is the main immunoglobulin in bovine mammary secretion products. US 2004/0167320 discusses a process and apparatus for separating molecules of interest from complex mixtures using improved methods of tangential flow filtration. Suitable molecules of interest, according to US 2004/0167320, include immunoglobulins. This US patent application contains an example of the purification of IgG1 from raw milk using diafiltration, wherein the process conditions and parameters are described in great detail. The separation of the much larger S-IgA immunoglobulins from milk is not disclosed however.
US 2003/0059512 discusses a method and apparatus for separation of milk and milk products involving one or more cross-flow filtration steps. In particular US 2003/0059512 suggests to separate defatted milk into a casein rich retentate fraction and a casein depleted permeate fraction, flowing said permeate to a subsequent cross-flow filtration module suitable to form a retentate fraction that is enriched with macromolecules such as albumin and immunoglobulins, which can be further separated and purified to form albumin and immunoglobulins using e.g. chromatography or cross-flow filtration. US 2003/0059512 does not contain any specific information or examples regarding the preparation of IgA, let alone S-IgA, enriched milk fractions.
U.S. Pat. No. 4,644,056 discusses a process of preparing a solution of lactic or colostric immunoglobulins by processing colostrums or milk. In accordance with this document colostrum is acidified to a pH of 4.0-5.5 and subjected to cross-flow filtration in a filtration unit with a mean pore size of 0.1-1.2 μm, whereafter the low-molecular weight components are removed by means of further cross-flow filtration in another filtration unit with a limit of separation of 5-80 kDa. The examples discuss diafiltration of the acidified colostrum using such filtration units, yielding solutions mainly containing IgG, although the presence of IgA, S-IgA and IgM in the solution was also reported using immunoelectrophoresis. Nevertheless, the process disclosed in U.S. Pat. No. 4,644,056 does not at all suffice for efficiently preparing S-IgA rich fractions from (non-colostrial or mature) milk in good yields.
Given the fact that there is a large difference in molecular weight as well as shape between IgA and IgG on the one hand and S-IgA on the other hand, the application of the prior art IgG-isolation processes for isolating S-IgA is not at all straight forward, let alone when efficient operation and sufficient yields are required, i.e. to render the method suitable for application in industrial scale production. As will be clear from the former, the need for a process that can be used to efficiently produce S-IgA rich (non-colostrial) milk fractions has not been satisfied by the prior art. It is an objective of the present invention to provide such a process.